抗人CD28单链抗体和嵌合抗体转移载体的构建及单链抗体基因在BmN细胞中的表达

发布时间：2018-01-08 07:10

本文关键词：抗人CD28单链抗体和嵌合抗体转移载体的构建及单链抗体基因在BmN细胞中的表达　出处：《苏州大学》2006年硕士论文　论文类型：学位论文

【摘要】： CD28分子是表达于T淋巴细胞表面的跨膜糖蛋白,参与介导T细胞活化所需的协同刺激信号,属于免疫球蛋白超家族。已有的研究表明,CD28单克隆抗体对肿瘤、自身免疫性疾病及移植排异等均具有重要的治疗意义,但体内长期使用会降低治疗效果。为了降低人抗鼠免疫反应,必须在临床应用前对CD28单克隆抗体进行改造。本研究在对抗人CD28单克隆抗体进行改造的基础上,成功构建了杆状病毒转移载体,并在BmN细胞中进行表达。主要成果如下: 1、将抗人CD28鼠源性单抗的可变区基因和人IgG1的恒定区基因,采用一种不需要限制性核酸内切酶和连接酶的新方法——三引物PCR(TP-PCR)法拼接成嵌合抗体基因,分别插入杆状病毒转移载体pAcUW3的ph和p10启动子后,成功构建了抗人CD28的重组转移载体pAcUW3/CD28-vH-γ_1,vL-κ。 2、采用TP-PCR法,将抗人CD28单抗的重、轻链可变区基因和人工设计的昆虫杆状病毒多角体蛋白基因偏好的连接肽序列,拼接成抗人CD28单链抗体基因,并将其插入昆虫杆状病毒转移载体pBacPAK8的ph基因启动子后,构建成重组转移载体pBacPAK8/CD28-ScFv。基因拼接时,在抗体链段的C端增加了6×His tag序列,便于表达产物的纯化。 3、采用脂质体介导的方法将重组转移载体pBacPAK8/CD28-ScFv和线性化的Bm-BacPAK6共转染BmN细胞,利用空斑分析以及蓝白斑筛选相结合的方法,筛选出重组杆状病毒Bm-BacPAK6 CD28-ScFv,并用PCR方法对此重组病毒进行了鉴定。 4、将重组病毒Bm-BacPAK6 CD28-ScFv接种BmN细胞,定期收取感染细胞,并对表达产物进行SDS-PAGE和Western Blotting分析。结果表明,在分子量约为28kD处有一清晰的亮带,说明抗人CD28单链抗体基因在BmN细胞中得到了特异性表达。表达始于24h,表达峰在72h。抗人CD28单链抗体和嵌合抗体基因转移载体的成功构建及抗人CD28单链抗体基因在BmN细胞中的表达为抗人CD28基因工程抗体的研制奠定了基础。
[Abstract]:CD28 molecules are transmembrane glycoproteins expressed on the surface of T lymphocytes. They are involved in the costimulatory signals required to mediate the activation of T cells and belong to the immunoglobulin superfamily. CD28 monoclonal antibody has important therapeutic significance for tumor, autoimmune disease and transplantation rejection, but long-term use in vivo will reduce the therapeutic effect. In order to reduce the human anti-mouse immune response. CD28 monoclonal antibody must be modified before clinical application. In this study, baculovirus transfer vector was successfully constructed on the basis of anti-human CD28 monoclonal antibody modification. And expressed in BmN cells. The main results are as follows: 1. The variable region gene of mouse monoclonal antibody against human CD28 and the constant region gene of human IgG1. A new method, which does not require restriction endonuclease and ligase, was used to splice the chimeric antibody gene into the chimeric antibody gene. After inserting ph and p10 promoters of baculovirus transfer vector pAcUW3, the recombinant transfer vector pAcUW3 / CD28-vH- 纬 1 was successfully constructed. VL- 魏. 2. Using TP-PCR method, the weight, light chain variable region gene of anti human CD28 monoclonal antibody and the preferentially linked peptide sequence of insect baculovirus polyhedrosis protein gene were sequenced. Splicing the single chain antibody gene against human CD28 and inserting it into the ph gene promoter of insect baculovirus transfer vector pBacPAK8. The recombinant transfer vector pBacPAK8 / CD28-ScFv. was constructed. When the gene was spliced, 6 脳 His tag sequence was added to the C end of the antibody chain, which was convenient for the purification of the expressed product. 3Recombinant transfer vector pBacPAK8/CD28-ScFv and linearized Bm-BacPAK6 were co-transfected into BmN cells by liposome-mediated method. The recombinant baculovirus Bm-BacPAK6 was screened by the combination of plaque analysis and blue and white spot screening. The recombinant virus was identified by PCR method. 4Recombinant virus Bm-BacPAK6 CD28-ScFv was inoculated into BmN cells and infected cells were collected regularly. The results of SDS-PAGE and Western Blotting analysis showed that there was a clear bright band at the molecular weight of about 28kD. The results showed that the anti-human CD28 single-chain antibody gene was specifically expressed in BmN cells, and the expression began at 24 h, and the expression peak was 72 h. Construction of anti-human CD28 single-chain antibody and chimeric antibody gene transfer vector and expression of anti-human CD28 single-chain antibody gene in BmN cells laid the foundation for the development of anti-human CD28 gene engineering antibody. Base.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：Q789;R392

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